MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation

Emerging evidence supports roles of enhancer RNAs (eRNAs) in regulating target gene. Here, we study eRNA regulation and function during skeletal myoblast differentiation. We provide a panoramic view of enhancer transcription and categorization of eRNAs. Master transcription factor MyoD is crucial in activating eRNA production. Super enhancer (se) generated seRNA-1 and -2 promote myogenic differentiation in vitro and in vivo. seRNA-1 regulates expression levels of two nearby genes, myoglobin (Mb) and apolipoprotein L6 (Apol6), by binding to heterogeneous nuclear ribonucleoprotein L (hnRNPL). A CAAA tract on seRNA-1 is essential in mediating seRNA-1/hnRNPL binding and function. Disruption of seRNA-1-hnRNPL interaction attenuates Pol II and H3K36me3 deposition at the Mb locus, in coincidence with the reduction of its transcription. Furthermore, analyses of hnRNPL binding transcriptome-wide reveal its association with eRNAs is a general phenomenon in multiple cells. Collectively, we propose that eRNA-hnRNPL interaction represents a mechanism contributing to target mRNA activation.

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Data analysis
Huating Wang Microscopy pictures were acquired with Leica microscope system DM 6000B; Victor X4 plate reader (Perkin Elmer) was used for luminescence assays; qPCR reactions were conducted using the Light-Cycler system (Roche Applied Science) All studies must disclose on these points even when the disclosure is negative.

Study description
Research sample (parameters: --very-sensitive --rdg 5,2 --score-min L,-0.6,-0.7) DREME in MEME suite version 4. The authors declare that data supporting the findings of this study are available within the article and Supplementary Information. GRO-seq, hnRNPL CLIP-seq and RNA-seq data reported in this paper were deposited in the Gene Expression Omnibus database under accession GSE114659.
No statistical test was used to determine sample size. Three biological replicates per group (detailed n is indicated in the figure legends) were collected to perform statistical testing. For GRO-seq, two biological replicates per group were used; for hnRNPL CLIP-seq, total RNA-seq, and polyA+ RNA-seq, one biological replicate were used.
No data were excluded. But during sequencing data analysis, standard quality control steps were applied to remove low-quality reads, reads aligning to the same coordinate. In the CLIP-seq analysis, a confident hnRNPL binding site was identified using FDR < 0.05 and supporting read count no less than 5 as the cutoff. In the GRO-seq analysis, to define putative eRNAs in MB and MT, transcripts overlapping with proteincoding genes, antisense transcripts, divergent transcripts and the other genic regions (rRNA, snRNA, miRNA, snoRNA, etc) were filtered and the remaining transcripts were defined as putative eRNAs.
All experimental data was repeated in multiple independent experiments as indicated in the figure legends and source data.
For all the animal experiments ( Fig. 1k, 5e-g, Supplementary Fig. 7h-m), mice were randomly allocated to distinct groups.
For immunofluorescence data collection (Fig. 3b, c, and Supplementary Fig. 4d, 5c, d, 7d, g, k-m, 11h, i), we performed the experiments in a blinded way. We randomly counted multiple fields per group and calculated the number of positively stained cells per field. Exact value can be found in source data. For other assays, we were not blinded to group allocation.
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